PCR is an efficient and cost effective way to copy or ‘amplify’ small segments of DNA or RNA. Using PCR, millions of copies of a section of DNA are made in just a few hours, yielding enough DNA required for analysis. This method allows clinicians to diagnose and monitor diseases using a minimal amount of sample, such as blood or tissue. Real-time PCR allows for amplification and detection to occur at the same time. One method of detection is done by utilizing oligonucleotide hydrolysis probes (also known as TaqMan® probes) having a fluorophore covalently attached, e.g., to the 5′ end of the oligonucleotide probe and a quencher attached, e.g., internally or at the 3′ end. Hydrolysis probes are dual-labeled oligonucleotide probes that rely on the 5′ to 3′ nuclease activity of Taq polymerase to cleave the hydrolysis probe during hybridization to the complementary target sequence, and result in fluorescent based detection.
Many real time PCR methods are currently limited by the fact that only one target nucleic acid sequence can be analyzed in a single closed reaction vessel, sometimes called the one-channel-one-target limitation. PCR detection methods that allow analysis of a greater number of targets in a single tube would provide additional capability and also provide more efficient detection testing methods. Thus, there is a need in the art for a quick and reliable method to detect two or more target nucleic acids in a single closed reaction, and the present disclosure provides benefits and solutions.